This invention relates to a mammalian cDNA which encodes a colon cancer marker and to the use of the cDNA and the encoded protein in the diagnosis and treatment of colon disorders, particularly colon cancer and polyps.
Phylogenetic relationships among organisms have been demonstrated many times, and studies from a diversity of prokaryotic and eukaryotic organisms suggest a more or less gradual evolution of molecules, biochemical and physiological mechanisms, and metabolic pathways. Despite different evolutionary pressures, the proteins of nematode, fly, rat, and man have common chemical and structural features and generally perform the same cellular function. Comparisons of the nucleic acid and protein sequences from organisms where structure and/or function are known accelerate the investigation of human sequences and allow the development of model systems for testing diagnostic and therapeutic agents for human conditions, diseases, and disorders.
Colorectal cancer is the fourth most common cancer and the second most common cause of cancer death in the United States with approximately 130,000 new cases and 55,000 deaths per year. Colon and rectal cancers share many environmental risk factors and both are found in individuals with specific genetic syndromes (Potter (1999) J Natl Cancer Institute 91:916-932). Colon cancer is the only cancer that occurs with approximately equal frequency in men and women, and the five-year survival rate following diagnosis of colon cancer is around 55% in the United States (Ries et al. (1990) National Institutes of Health, DHHS Publ. No. (NI)90-2789).
Colon cancer is causally related to both genes and the environment. Several molecular pathways have been linked to the development of colon cancer, and the expression of key genes in any of these pathways may be affected by inherited or acquired mutation or by hypermethylation. There is a particular need to identify genes for which changes in expression may provide an early indicator of colon cancer or a predisposition for the development of colon cancer.
For example, it is well known that abnormal patterns of DNA methylation occur consistently in human tumors and include, simultaneously, widespread genomic hypomethylation and localized areas of increased methylation. In colon cancer in particular, it has been found that these changes occur early in tumor progression such as in premalignant polyps that precede colon cancer. Indeed, DNA methyltransferase, the enzyme that performs DNA methylation, is significantly increased in histologically normal mucosa from patients with colon cancer or in the benign polyps that precede cancer. This increase continues during the progression of colonic neoplasms (El-Deiry et al. (1991) Proc Natl Acad Sci USA 88:3470-3474). Increased DNA methylation occurs in G+C rich areas of genomic DNA termed xe2x80x9cCpG islandsxe2x80x9d that are important for maintenance of an xe2x80x9copenxe2x80x9d transcriptional conformation around genes, and hypermethylation of these regions results in a xe2x80x9cclosedxe2x80x9d conformation that silences gene transcription. It has been suggested that the silencing or downregulation of differentiation genes by such abnormal methylation of CpG islands may prevent differentiation in immortalized cells (Antequera et al. (1990) Cell 62:503-514).
Familial adenomatous polyposis (FAP) is a rare autosomal dominant syndrome that precedes colon cancer and is caused by an inherited mutation in the adenomatous polyposis coli (APC) gene. FAP is characterized by the early development of multiple colorectal adenomas that progress to cancer at a mean age of 44 years. The APC gene is a part of the APC-xcex2-catenin-Tcf (T-cell factor) pathway. Impairment of this pathway results in the loss of orderly replication, adhesion, and migration of colonic epithelial cells that results in the growth of polyps. A series of other genetic changes follow activation of the APC-xcex2-catenin-Tcf pathway and accompanies the transition from normal colonic mucosa to metastatic carcinoma. These changes include mutation of the K-Ras proto-oncogene, changes in methylation patterns, and mutation or loss of the tumor suppressor genes p53 and Smad4/DPC4. While the inheritance of a mutated APC gene is a rare event, the loss or mutation of APC and the consequent effects on the APC-xcex2-catenin-Tcf pathway is believed to be central to the majority of colon cancers in the general population.
Hereditary nonpolyposis colorectal cancer (HNPCC) is another inherited autosomal dominant syndrome with a less well defined phenotype than FAP. HNPCC, which accounts for about 2% of colorectal cancer cases, is distinguished by the tendency to early onset of cancer and the development of other cancers, particularly those involving the endometrium, urinary tract, stomach, and biliary system. HNPCC results from the mutation of one or more genes in the DNA mis-match repair (MMR) pathway. Mutations in two human MMR genes, MSH2 and MLH1, are found in a large majority of HNPCC families identified to date. The DNA MMR pathway identifies and repairs errors that result from the activity of DNA polymerase during replication. Furthermore, loss of MMR activity contributes to cancer progression through accumulation of other gene mutations and deletions, such as loss of the BAX gene which controls apoptosis, and the TGFxcex2 receptor II gene which controls cell growth. Because of the potential for irreparable damage to DNA in an individual with a DNA MMR defect, progression to carcinoma is more rapid than usual.
Although ulcerative colitis is a minor contributor to colon cancer, affected individuals have about a 20-fold increase in risk for developing cancer. Progression is associated with mutations in the p53 gene which may occur early, appearing even in histologically normal tissue. The progression of the disease from ulcerative colitis to dysplasia/carcinoma without an intermediate polyp state suggests a high degree of mutagenic activity resulting from the exposure of proliferating cells in the colonic mucosa to the colonic contents.
Almost all colon cancers arise from cells in which the estrogen receptor (ER) gene has been silenced. The silencing of ER gene transcription is age related and linked to hypermethylation of the ER gene (Issa et al. (1994) Nature Genetics 7:536-540). Introduction and expression of an exogenous ER coding sequence into cultured colon carcinoma cells results in marked suppression of growth. Inhibition of cancer cell invasion depends on the function of the hormone binding domain and the N-terminal zinc finger region of the ER (Platet et al. (2000) Mol Endocrinol 14:999-1009). Activation of the ER by hormone binding induces transcription of specific target genes and may be linked to the reduction in cancer invasiveness. In the absence of hormone, protein-protein interactions with the zinc finger region may also contribute to the inhibition of cancer cell migration. The connection between loss of the ER protein in colonic epithelial cells and the consequent development of cancer has not been established.
The FYVE-finger proteinsplay roles in cellular processes such as receptor signaling, vesicular trafficking, and actin-regulated membrane rearrangements (Stenmark and Aasland (1999) J Cell Science 112:4175-4183). The FYVE domain is a type of zinc finger that typically contains eight conserved cysteines which bind two Zn2+ cations, has conserved glycine and arginine residues, and a basic motif with the consensus sequence R(R/K)HHCR. The FYVE domain binds to phosphoinositides found in specific membranes. The presence of other domains in FYVE-finger proteins may mediate proteinxe2x80x94protein interactions with other molecules at the membrane and may be involved in the recruitment of signaling molecules such as small GTPases to membranes at particular cellular locations. In mice with a null muation in the FYVE finger protein Hrs, embryos showed defects in ventral folding morphogenesis and died in utero (Komada and Soriano (1999) Genes Dev 13:1475-1485). The embryos developed with their ventral region outside of the yolk sac, had two independent bilateral heart tubes, and no foregut. Mutations in genes that control cellular differentiation and proliferation in the intestine may be linked with colon disease (Dove et al. (1998) Phil Trans R Soc Lond B 353:915-923).
Clearly there are a number of genetic alterations associated with colon cancer and with the development and progression of the disease. Particularly, downregulation of expression or deletion of genes potentially provide early indicators of cancer development, and may also be used to monitor disease progression or provide possible therapeutic targets. The specific genes affected in a given case of colon cancer depend on the molecular progression of the disease. Identification of additional genes associated with colon cancer and the precancerous state would provide more reliable diagnostic patterns associated with the development and progression of the disease.
The discovery of a mammalian cDNA encoding a colon cancer marker satisfies a need in the art by providing compositions which are useful in the diagnosis and treatment of colon disorders, particularly colon cancer and polyps.
The invention is based on the discovery of a mammalian cDNA which encodes a colon cancer marker (CCM), which is useful in the diagnosis and treatment of colon disorders, particularly colon cancer and polyps.
The invention provides an isolated mammalian cDNA or a fragment thereof encoding a mammalian protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO:1, a variant having at least 85% identity to the amino acid sequence of SEQ ID NO:1, an antigenic epitope of SEQ ID NO:1, and a biologically active portion of SEQ ID NO:1. The invention also provides an isolated mammalian cDNA or the complement thereof selected from the group consisting of a nucleic acid sequence of SEQ ID NO:2, a variant having at least 87% identity to the nucleic acid sequence of SEQ ID NO:2, a fragment of SEQ ID NOs:3-22, an oligonucleotide of SEQ ID NOs:2-34. The invention additionally provides a composition, a substrate, and a probe comprising the cDNA, or the complement of the cDNA, encoding CCM. The invention further provides a vector containing the cDNA, a host cell containing the vector and a method for using the cDNA to make CCM. The invention still further provides a transgenic cell line or organism comprising the vector containing the cDNA encoding CCM. The invention additionally provides a mammalian fragment or the complement thereof selected from the group consisting of SEQ ID NOs:23-34. In one aspect, the invention provides a substrate containing at least one of these fragments. In a second aspect, the invention provides a probe comprising the fragment which can be used in methods of detection, screening, and purification. In a further aspect, the probe is a single stranded complementary RNA or DNA molecule.
The invention provides a method for using a cDNA to detect the differential expression of a nucleic acid in a sample comprising hybridizing a probe to the nucleic acids, thereby forming hybridization complexes and comparing hybridization complex formation with a standard, wherein the comparison indicates the differential expression of the cDNA in the sample. In one aspect, the method of detection further comprises amplifying the nucleic acids of the sample prior to hybridization. In another aspect, the method showing differential expression of the cDNA is used to diagnose colon disorders, particularly colon cancer and polyps. In another aspect, the cDNA or a fragment or a complement thereof may comprise an element on an array.
The invention additionally provides a method for using a cDNA or a fragment or a complement thereof to screen a library or plurality of molecules or compounds to identify at least one ligand which specifically binds the cDNA, the method comprising combining the cDNA with the molecules or compounds under conditions allowing specific binding, and detecting specific binding to the cDNA, thereby identifying a ligand which specifically binds the cDNA. In one aspect, the molecules or compounds are selected from aptamers, DNA molecules, RNA molecules, peptide nucleic acids, artificial chromosome constructions, peptides, transcription factors, repressors, and regulatory molecules.
The invention provides a purified mammalian protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO:1, a variant having 85% identity to the amino acid sequence of SEQ ID NO:1, an antigenic epitope of SEQ ID NO:1, and a biologically active portion of SEQ ID NO:1. The invention also provides a composition comprising the purified protein or a portion thereof in conjunction with a pharmaceutical carrier. The invention further provides a method of using the CCM to treat a subject with colon disorders, particularly colon cancer and polyps comprising administering to a patient in need of such treatment the composition containing the purified protein. The invention still further provides a method for using a protein to screen a library or a plurality of molecules or compounds to identify at least one ligand, the method comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein. In one aspect, the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs. In another aspect, the ligand is used to treat a subject with colon disorders, particularly colon cancer and polyps.
The invention provides a method of using a mammalian protein to screen a subject sample for antibodies which specifically bind the protein comprising isolating antibodies from the subject sample, contacting the isolated antibodies with the protein under conditions that allow specific binding, dissociating the antibody from the bound-protein, and comparing the quantity of antibody with known standards, wherein the presence or quantity of antibody is diagnostic of colon disorders, particularly colon cancer and polyps.
The invention also provides a method of using a mammalian protein to prepare and purify antibodies comprising immunizing a animal with the protein under conditions to elicit an antibody response, isolating animal antibodies, attaching the protein to a substrate, contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein, dissociating the antibodies from the protein, thereby obtaining purified antibodies.
The invention provides a purified antibody which binds specifically to a protein which is expressed in colon disorders, particularly colon cancer and polyps. The invention also provides a method of using an antibody to diagnose colon disorders, particularly colon cancer and polyps comprising combining the antibody comparing the quantity of bound antibody to known standards, thereby establishing the presence of colon disorders, particularly colon cancer and polyps. The invention further provides a method of using an antibody to treat colon disorders, particularly colon cancer and polyps comprising administering to a patient in need of such treatment a pharmaceutical composition comprising the purified antibody.
The invention provides a method for inserting a marker gene into the genomic DNA of a mammal to disrupt the expression of the endogenous polynucleotide. The invention also provides a method for using a cDNA to produce a mammalian model system, the method comprising constructing a vector containing the cDNA selected from SEQ ID NOs:2-34, transforming the vector into an embryonic stem cell, selecting a transformed embryonic stem, microinjecting the transformed embryonic stem cell into a mammalian blastocyst, thereby forming a chimeric blastocyst, transferring the chimeric blastocyst into a pseudopregnant dam, wherein the dam gives birth to a chimeric offspring containing the cDNA in its germ line, and breeding the chimeric mammal to produce a homozygous, mammalian model system.